Deallergenization and detoxification of castor bean pomace



United States Patent 3 101 266 DEALLERGENTZATICN AND DETOXIFICATHON OFCAST OR BEAN POMACE Joseph R. Spies, Arlington, Va, Emery J. Coulson,.

Takoma Park, Md., and Percy A. Wells, Abington, Pa., assignors to theUnited States of America as represented by the Secretary of AgricultureNo Drawing. Filed Sept. 13, 1960, Ser. No. 55,809

7 Claims. (Cl. 71-23) (Granted under Title 35, U.S. Code (1952), see.266) ception-ally stable allergenic component. ln processing the castorbean for castor oil these components are retained in the pomace, theground, defatted bean residue. The toxicity of ricin is known to bedestroyed when heated in water to the boilingpoint or even to thecoagulation temperature of the protein. Despite intensive ellorts toinactivate the allergen, the presence of this factor in castor beanpomace is still a serious problem in handling castor bean pomace orproducts into which the pomace is incorporated. This is because theallergen has unusually potent sensitizing capacity for those exposed tothe dust of castor bean pomace. Even trace amounts provoke severe asthmain hypersensitive individuals. Although processes have been proposed fordeactivating the allergen, none of these currently reported sulfice toyield a product which can be safely handled by all people.

An object of the present invention is to render castor bean pomacenonallergenic and nontoxic. A further object is to provide a castor beanproduct having both organic and inorganic nutritive values as afertilizer. Still another object is to provide a fertilizer containingcastor bean pomace which can be used without endangering the health ofhandlers and consumers.

9 According to the present invention, castor bean pomace, water andcalcium hydroxide are combined to give a mixture having a pH in therange of about 9.5 to 12.5, and the alkaline mixture is heated at about100 to 120 C. for a time interval of about eight to sixty minutes. Thistreatment inactivates the allergen and destroys the toxic components.The pomace product is used as an alkaline fertilizer, or preferably thereaction mixture is partially or completely neutralized to give apomace-calcium phosphate solid, a new combination fertilizer.

Various alternatives are available for handling the products asfertilizers. For local use, the wet slurry may be directly applied tofields, or solids may be collected from the slurry by conventionalmeans, such as centrifuging or filtering, and the product used as a wetsludge. in practice, it is anticipated that a considerable portion orall of the slurry or the collected pomace product will be dried, thusfacilitating storage, shipping and consumer use.

The particular means of combining the pomace, water and calciumhydroxide to achieve the desired pH is imj A convenient procedure is tocombine calcium hydroxide and pomace on a percentage Q material to theinvention.

basis; for example, eight percent calcium hydroxide, dryweight basis,and add calculated amounts of water. Alternatively, a freshly preparedaqueous suspension of calcium hydroxide of known concentration can becombined with dry pomace or with an aqueous slurry of pomace.

In preparing the pomace-calcium phosphate fertilizer,

the addition of one mole of phosphoric acid for each mole of calciumhydroxide combined with the pomace gives a product with a pH in therange of 5.5 to 6.5. A neutral (pH 7.0) or slightly alkaline fertilizermay be produced, if desired, by adding less phosphoric acid to thealkaline pomace mixture. Forconvenience and safety, we allowed theheated mixture to cool before adding the phosphoric acid, but this isnot a requirement of the process.

.It has previously been established that the toxic and allergenicprinciples are water soluble, although it is not feasible to remove allthe allergen from the pomace by water leaching. Accordingly, forpurposes of accuracy and of controlled analysis, destruction of thetoxic and allergenic components in the pomace by the inventive processwas determined by tests made on the aqueous solution separated fromtreated and control pomace slurries. I

In many, instances the inventive process is more convenientlydemonstrated by application to isolated castor bean allergen. Theisolation of castor bean allergen was described in J. Am. Chem. Soc.,65, 1720 1943), the fraction being called CB-lA. Dialysis of CB-lA withsuccessive changes of water gives a further purified castor beanallergen, (CB-1A)E, which is nontoxic and is not coagulated attemperature up to 170 0., either at neutrality or at pH values above 12.The use of (CB-1A)E rather than pomace in tests on humans eliminates thepossibility of toxic effects.

Toxicity was determined by subcutaneous injection in guinea pigs of oneml. of treated or untreated pomace solution. 'Iwofold serial dilutionswere injected through a maximum dosage of extract from 12.8 mg. ofcastor bean pomace. The average lethal dose of untreated pomace was 0.1mg. per 3001-20 g. guinea pig, the range being from 0.05 to 0.2 mg.Results of toxicity tests are described (cf. Table I) as follows: NS, nosymptoms with the quantity of pomace indicated in mgs.; I, induration atthe site of injection, but no deaths or necrosis; and N, no deaths with12.8 mgs. of pomace, but necrosis at the site of injection withindicated quantity of pomace and no symptoms with smaller amounts.

Precipitin tests employed clarified rabbit antiserum obtained fromrabbits immunized to the principal antigen of (CB-1A) E.Fifteen-hundredths milliliter of clarified antiserum in a 5 x 45 mm.tube was mixed with 0.15 ml. of test solution and incubated 30 minutesat 37 C. The tubes were placed at 5 C. and the preciptate read visuallyafter 24 hours. Dilutions were made with a buttered saline solution, pH7.0. Treated pomace and (CB-1A)E solutions were tested at 1:10 dilutionwith respect to their original (CB-1A)E content to determine destructionof preciptating capacity. Absence of a precipitate with this test showsdestruction of at least 98 percent of the precipitating capacity.

The Prausnitz-Kustner or passive transfer test on humans was used toevaluate loss of allergenic properties of the castor bean extracts.Essentially, the same method was used as described in Annals of Allergy,18, 393 The recipients were free from antihistamine medication. A zeroallergenic property was recorded only'when the test showed that 99.95percent of the allergen had been destroyed. Determination of this degreeof completeness of destruction of the allergen was necessary because ofthe extremely small amount of castor bean allergen capable of provokingan asthmatic attack in a hypersensitive person.

The castor bean pomace for the examples was prepared from castor beansby decortification, defatting with ether, and grinding to pass a 40-meshsieve.

d Our invention is illustrated but not limitedby the following examples:

EXAMPLE 1 To 78 mg. samples of defatted, powdered castor bean pomacewere added calculated amounts of water and. an aqueous calcium hydroxidesuspension to give a total volume of 3.0 ml. and percentages of calciumhydroxide, based on pomace, of 0, 1, 2, 4, 8, and 16. The ingredientswere mixed and heated in closed tubes for one hour at temperatures of60, 80 and 100 C. The mixtures were cooled to room temperature and thencala,1o1,ees

Table 1 EFFECT OF HEATING OASTOR BEAN POMACE WITH AQUEOUS CALCIUMHYDROXIDE N ALLERGEN AND TOXIC COMPONENTS Temperature Ca(OH)z I Aller-Aller- M b PerpH Precipgenie ToxicpH d Preeipgenie Toxiceent 0 itin Bpropity B itin propity B erty i erty 1 0 0 6. 2 4+ 0.8 N 6. 4 4+ 12.8 NS0.0035 1 7. 7 4+ 12. 8 I 7. 4+ 12. 8 NS 0.007 2 9. 5 3+ 12.8 I 9.0 1+12.8 NS 0. 014 4 10. 4 1+ 12. 8 I 10. 3 1+ 12. 8 I 0.028 8 .11. 8 0 12.8I 11.8 0 0 12.8 I 0.056 16 12.5 0 12. 8 I 12.3 0 0 12.8 I

n Heated one hour at indicated temperature, centigrade. b Molarity. nPercent calcium hydroxide based on weight of pomace. d pH oi suspensionafter heating and cooling. Antigenic or immune precipitating property ofcastor bean allergen. f As determined on (CB-1.4.)E under correspondingconditions. s Described in text.

culated amounts of Water and 0.1 M o-phosphoric acid EXAMPLE 2 wereadded (one mole of phosphoric acid for each mole of calcium hydroxide)so that the total volume of the slurry was 6.0 ml. The pH of theneutralized slurry Was in the range of 5.5 to 6.5. After standingseveral minutes, each slurry was centrifuged and the supernatantsolution separated from the pomace-calcium phosphate solids. Eachsupernatant solution was tested as previously described. Heating at C.was not suflicient to destroy toxicity at most levels of pH and resultsat this temperature are omitted from the summary presented in Table I.

The combination of heating a solution of pH 11.8 for one hour at 100 C.deactivated both the antigenic and allergenic properties of the castorbean allergen. Lowering the temperature to C., same pH and time,destroys only the antigenic property. Elimination of ricin toxicity wasconfirmed. The induration at the injection Thirty mg. samples of (CB1A)Ewere dissolved in the calculated volumes of water and a freshly preparedaqueous suspension of calcium hydroxide or 10 rug/ml.) was added to givea total volume of 3.0 ml. and molar concentrations of calcium hydroxideof 0, 0.0035, 0.007, 0.014, 0.028 and 0.056. The solutions orsuspensions were heated in sealed tubes at 80, 100 and C., cooled andthe pH determined. Excess calcium hydroxide was neutralized with 0.1 Mo-phosphoric acid, mole for mole, and the final volume was adjusted to6.0 ml. with water. The p-H of resulting solutions or suspensions was6105. After occasional shaking for several minutes, the precipitate ofcalcium acid phosphate, if any, was separated by centrifuging. The clearsolutions or their dilutions were used for precipitin and allergentests. Results are summarized in Table II.

Table II EFFECT OF HEATING OASTOR BEAN ALLERGEN (CB1A)E, WITH AQUEOUSCALCIUTM HYDROXIDE ON ITS IMMUNE PRECIPITATING CAPACITY AND ON ANALLERGENIC PROPERTY (REAGIN NEUTRALIZATION) Temperature Ca(OE)z, 80 100120 pH Precip- Allergenie pH Precip- Allergenic pH Precin- Allergenicitin d property itin d property 3 itin d property Heated one hour atindicated temperature, C.

b Molarity.

* pH of solution or suspension after heating and cooling, beforeaddition of phosphoric acid.

d Preeipitin reaction with 1:10 dilution of (OB1A)E.

e 0, indicates destruction of 99.95% or more reagin neutralizationcapacity of (CB1A)E; indicates less than 99.95% destruction of reaginneutralization capacity of (CB1A)E.

As shown in Table II, at 80 C. :for one hour, the antigenic property(immune precipitating capacity) of the castor bean allergen wasdestroyed at pH 11.9, but the allergenic property (reagin neutralizingproperty) was not destroyed even at pH 12.5. At 100 and 120 C., theprecipitating capacity was destroyed at pH values of 10.8 and 9.8 andthe reagin neutralizing property at pH values of 12.0 and 8.7,respectively. The combinations of either pH about 12 and 100 C. or pHabout 9.5 and 120 C. :for one hour suflice to destroy both properties ofthe castor bean allergen.

EXAMPLE 3 This example varied fromExample 2 in that all samples wereprepared with 8 percent (0.028 M) calcium hydroxide and that the tubeswere heated at 100 and,

120 'C. for periods of time ranging from zero (control) to 64 minutes.The rates of destruction of antigenic and allergenic properties areshown in Table 111.,

These results show that at pH about 12.5 the castor bean allergen isdeactivated in about 30 minutes at 100 C. and in eight minutes or lessat 120 C.

Table III EFFECT OF TIME OF HEATING CASTOR BEAN ALLERGEN (CB1A)E', WITH0.028 MOLAR AQUEOUS CALCIUM HYDROXI E ON ITS IMMUNE PRECIPITATIN GCAPACITY AND ON AN ALLERGENIC PROPERTY (REAGIN NEUTRALIZATION)Temperature 100 120 Time, minutes Aller- AllerpH B Precipgenie pHPrecipgenie itin b propitin property erty pH of solution or suspensionafter heating and cooling, before addition of phosphoric acid.

b Precipitin reaction with 1:10 dilution of (OB1A)E.

0, indicates destruction of 99.95% or more reagin neutralizationcapacity of (CB1A)E; indicates less than 99.95% destruction of reaginneutralization capacity of (OB1A)E.

castor bean allergen. Addition of phosphoric acid results in reactionwith the calcium hydroxide in the mixtureand forms calciumsuperphosphate, which in itself is an extensively used fertilizer. Thecalcium superphosphate-in'activated poma'ce mixture is, therefore, aharmless- 2. The process of claim 1 in which the pH of the alkalinemixture is about 9.5 andthe heating is at 120 C. for about one hour. I

3. The process of claim 1 in which the pH of the alkaline mixture isabout 12.5 and the heating is at 120 C. for about eight minutes. I

, 4. The process of claim 1 in which the pH of the alkaline mixture isabout 12.5 and the heating is at C. for about 30 minutes.

5. The process of claim 1 in which the pH of the alkaline mixture isabout 12.0 and the heating is at 100 C. for about one hour.

6. A process comprising combining castor bean pomace,

water, and calcium hydroxide togive a mixture having a 1 pH in the rangeof about 9.5 to about 12.5, heating the alkaline mixture at about C. to100 C. for about eight to sixty minutes to inactivate the castor beanallergen and destroy toxic components in the pornace, and addingo-phosphoric acid to give a harmless fertilizer of calciumsuperphosphate and inactivated castor bean pomace which possesses highplant nutrient value.

'7. The process of claim 6 in which the phosphoric acid is added on amole for mole basis calculated on the calcium hydroxide added to thepomace.

References Cited in the file of this patent UNITED STATES PATENTS OTHERREFERENCES Gardner et al.: The Journal of the American Oil ChemistsSociety, vol. 37, #3, March 1960, pages 142.- 148 (paper presented atfall meeting, American Oil Chemists Society, Los Angeles, Calif,September 28-30,

1. A PROCESS COMPRISING COMBINING CASTOR BEAM POMACE, WATER, AN CALCIUMHYDROXIDE TO GIVE A MIXTURE HAVING A PH IN THE RANGE OF ABOUT 9.5 TOABOUT 12.5, AND HEATING THE ALKAINE MIXTURE AT ABOUT 120*C. TO 100*C.FOR A TIME INTERVAL OF ABOUT EIGHT TO SIXTY MINUTES TO INACTIVATE THECASTEOR BEAN ALLERGEN AND DESTROY THE TOXIC COMPONENTS IN THE POMACE.